Project Summary/Abstract Natural biological circuits are far superior in regulated performance when compared to current synthetic designs, and this gap will always be wide without a much stronger set of theoretical rules. Though many designs and interesting functions have been demonstrated, there is a fundamental noise issue that prevents reliable, predictable performance of synthetic biological circuits ? and therefore use in any promising end applications. Toward identifying fundamental noise control principles, the goal here is to build a synchronized circadian clock that can be coupled to other functions in gut bacteria, such as drug delivery. Cyanobacterium Synechococcus elongatus is the simplest organism with a known circadian clock, which regulates many of the organism's functions in a 24-hour cycle. The molecular basis for this circadian clock is an oscillating phosphorylation cycle of protein KaiC. This cyanobacterial clock had been crudely transplanted in E. coli, a gut bacterium without a natural circadian system, but performed very poorly because of severe noise issues. To improve upon that effort, the circadian clock will be rebuilt in E. coli with its naturally robust control strategies of (1) protein-phosphorylation and (2) transcription-translation cycle control loops. Since some cyanobacterial parts are incompatible in E. coli and other therapeutic gut bacteria, some feedback strategies will be introduced in principle/behavior using other parts, such as heterologous repressors and relief to tune production amounts and timing of system components. An oscillating function amongst cells, such as therapeutic bacteria secreting a given daily drug dosage, would be most effective with a synchronized population. While oscillator circuits have shown ?generational? synchronicity based on cell division cycles, those cells were not truly synchronized without regulating external stimuli, as may be the case in the human gut. The second main goal is to couple a quorum sensing system with an oscillator like the circadian clock. This further enhances the control theory platform by adding a cell-to-cell communication dimension, which has been largely underexplored for synthetic circuits. Assembling these control circuits will serve as testing grounds for control theories and mathematical models, hopefully identifying general principles or fundamental rules for controlling biological noise that will be applicable to all synthetic circuits ? which will be a huge advancement toward creating mature engineered cells for real applications.